Running title: Negative regulation of OsBSK3 via its …...2015/12/22 · 21 Baowen Zhang1,...

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Running title 1

Negative regulation of OsBSK3 via its TPR domain 2

3

4

Corresponding Author 5

Dr Wenqiang Tang 6

College of Life Sciences 7

Hebei Normal University 8

Shijiazhuang Hebei Province 9

China 10

Email tangwqmailhebtueducn 11

Phone 86-311-80787594 12

13

Research Area 14

Signaling and Response 15

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Plant Physiology Preview Published on December 23 2015 as DOI101104pp1501668

Copyright 2015 by the American Society of Plant Biologists

httpsplantphysiolorgDownloaded on December 15 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

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OsBRI1 activates BR signaling by preventing binding between the TPR and kinase 18

domains of OsBSK3 via phosphorylation 19

20

Baowen Zhang1 Xiaolong Wang1 Zhiying Zhao1 Ruiju Wang1 Xiahe Huang2 Yali Zhu1 21

Li Yuan1 Yingchun Wang2 Xiaodong Xu1 Alma L Burlingame3 Yingjie Gao1 Yu Sun1 22

and Wenqiang Tang1 23

24

25

1 Key Laboratory of Molecular and Cellular Biology of Ministry of Education Hebei 26

Collaboration Innovation Center for Cell Signaling Hebei Key Laboratory of 27

Molecular and Cellular Biology College of Life Sciences Hebei Normal University 28

Shijiazhuang 050016 China 29

2 Key Laboratory of Molecular Development Biology Insitute of Genetics and 30

Development Biology Chinese Academy of Sciences Beijing 100101 China 31

3 Mass spectrometry facility Department of Pharmaceutical Chemistry University of 32

California San Francisco California 94143 33

34

35

36

37

38

One-sentence summary 39

OsBRI1 disrupts the interaction between the TPR and kinase domains of OsBSK3 via 40

phosphorylation this activates BR signaling by increasing the binding between OsBSK3rsquos 41

kinase domain and BSU1 42

43

44


3

Financial support 45

This study was supported by grants from the National Natural Science Foundation of 46

China (31071246 and 2014CB943404) the Department of Education of Hebei Province 47

China (CPRC035 and LJRC025) and the Scientific Research Foundation for the 48

Returned Overseas Chinese Scholars Hebei Province China (20100702) 49

50

Address correspondence to tangwqmailhebtueducn (WT) 51

52

AUTHOR CONTRIBUTIONS 53

WT conceived the project and designed the experiment BZ did most of the experiments 54

and analysis presented in this study XW generated the point mutation OsBSK3 forms 55

and the corresponding transgenic plants ZZ helped all the rice related phenotype 56

analysis RW helped generating N390-S215E and N390 overexpression transgenic 57

d61-2 and WT rice plants XH YW and ALB involved in identification of 58

phosphorylation sites on OsBSK3 and AtBSK3 by mass spectrometry YZ helped 59

generating figure 4B 4C and 4D LY and XX helped the firefly luciferase 60

complementation analysis YG provided technical assistance to BZ WT BZ and YS 61

wrote the paper together 62

63

64

65


4

ABSTRACT 66

Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases 67

(RLCKs) however the molecular mechanisms that create an effective on-off switch are 68

unknown The receptor kinase BRI1 transduces brassinosteroid (BR) signal by 69

phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs which 70


5

contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain Here 71

we show that the BR signaling function of BSKs is conserved in Arabidopsis and rice 72

and that the TPR domain of BSKs functions as a ldquophospho-switchablerdquo autoregulatory 73

domain to control BSKsrsquo activity Genetic studies revealed that OsBSK3 is a positive 74

regulator of BR signaling in rice while in vivo and in vitro assays demonstrated that 75

OsBRI1 interacts directly with and phosphorylates OsBSK3 The TPR domain of 76

OsBSK3 which interacts directly with the proteinrsquos kinase domain serves as an 77

autoinhibitory domain to prevent OsBSK3 from interacting with BSU1 Phosphorylation 78

of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains 79

thereby increasing the binding between OsBSK3rsquos kinase domain and BSU1 Our results 80

not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in 81

rice but also provide insight into the molecular mechanism by which BSK family 82

proteins are inhibited under basal conditions but switched on by the upstream receptor 83

kinase BRI1 84

85


6

INTRODUCTION 86

Brassinosteroids (BRs) are a group of natural polyhydroxy steroids that regulate diverse 87

physiological processes in plants including growth promotion skotomorphogenesis 88

organ boundary formation stomata development sex determination vascular 89

differentiation male fertility seed germination flowering senescence and resistance to 90

various abiotic and biotic stresses (Gendron et al 2012 Gomes 2011 Hartwig et al 91

2011 Kim et al 2012 Kutschera and Wang 2012) Over the past decade using 92

Arabidopsis thaliana as a model plant genetic biochemical molecular and proteomic 93

studies have revealed details about the BR signal transduction mechanisms from 94

perception of the signal by the receptor kinase BR INSENSITIVE 1 (BRI1) to 95

downstream transcriptional regulation The BR signaling pathway in Arabidopsis 96

represents one of the best-characterized receptor kinase pathways in plants and therefore 97

serves as a model for understanding receptor kinase signaling in general and for 98

understanding BR signaling in crops 99

BRs are recognized by a membrane-localized leucine-rich repeat receptor-like kinase 100

BRI1 and its co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) (Li and 101

Chory 1997 Santiago et al 2013 Sun et al 2013) BR binding promotes the 102

association of BRI1 with BAK1 and enables trans-phosphorylation between the 103

cytoplasmic kinase domains of the two receptors (Li et al 2002 Nam and Li 2002 104

Wang et al 2005 2008) BRI1 then phosphorylates two membrane-localized 105

receptor-like cytoplasmic kinases (RLCKs) BR SIGNALING KINASES (BSKs) and 106

CONSTITUTIVE DIFFERENTIAL GROWTH 1 (CDG1) leading to activation of the 107

protein phosphatase bri1-SUPPRESSOR 1 (BSU1) (Kim et al 2009 2011 Tang et al 108

2008) BSU1 dephosphorylates and inhibits the GSK3Shaggy-like kinase BR 109

INSENSITIVE 2 (BIN2) (Kim et al 2009) In the absence of BRs BIN2 phosphorylates 110

BRASSINAZOLE RESISTANT 1 (BZR1) family transcription factors preventing them 111

from regulating the transcription of downstream targets (He et al 2002 2005 Vert and 112


7

Chory 2006) BR signaling inhibits BIN2 and allows BZR1 to be dephosphorylated by 113

PROTEIN PHOSPHATASE 2A (PP2A) (Tang et al 2011) Together with their binding 114

partners dephosphorylated BZR1 family transcription factors bind to BR response 115

element (BRRE) or E-box cis element and regulate the expression of many 116

BR-responsive genes (He et al 2005 Yin et al 2005 Sun et al 2010) 117

The interaction with a membrane-localized receptor-like kinase is not unique for BSKs 118

and CDG1 it has also been observed for a number of other RLCKs including M-LOCUS 119

PROTEIN KINASE (Kakita et al 2007) BOTRYTIS-INDUCED KINASE 1 (Lu et al 120

2010) CAST AWAY (CST) (Burr et al 2011) OsRLCK185 (Yamaguchi et al 2013) 121

and AVRPPHB SUSCEPTIBLE-LIKE 1 (Zhang et al 2010) Therefore interacting with 122

a receptor-like kinase to regulate cellular signaling pathways might represent a general 123

function of RLCKs While many RLCKs regulate cellular responses by phosphorylating 124

one or more substrates directly there are many RLCKs encoded in the Arabidopsis 125

genome (eg BSKs) that contain a kinase domain that lacks conserved amino acids 126

believed to be essential for ATP binding or phosphoryl transfer (Castells and Casacuberta 127

2007 Roux et al 2014) The molecular mechanisms by which these atypical kinases 128

switch signaling pathways on and off to regulate cellular functions remain to be explored 129

In this study we demonstrate that OsBSK3 plays a conserved role in transducing BR 130

signal from OsBRI1 to downstream components in rice Similar to Arabidopsis BSKs 131

(AtBSKs) OsBSK3 contains a kinase domain and a C-terminal tetratricopeptide repeat 132

(TPR) domain Our data show that the TPR domain of OsBSK3 interacts directly with the 133

proteinrsquos kinase domain and that it serves as an autoinhibitory domain to prevent 134

OsBSK3 from interacting with BSU1 Phosphorylation of OsBSK3 by OsBRI1 is critical 135

for the regulation of BR signaling because it prevents binding between the TPR and 136

kinase domains of OsBSK3 thus enabling binding between the kinase domain of 137

OsBSK3 and BSU1 138

139


8

RESULTS 140

Rice BSK3 positively regulates BR signaling in Arabidopsis 141

Previously it was shown that AtBSKs mediate BR signal transduction from the 142

membrane receptor AtBRI1 to the downstream phosphatase AtBSU1 (Kim et al 2009) 143

To investigate whether a similar mechanism exists in rice a BLAST search against the 144

rice genome was conducted using twelve AtBSK protein sequences Four AtBSK 145

orthologs were identified in rice Based on their sequence homology to AtBSK3 the 146

orthologs were named OsBSK1 (Os03g04050) OsBSK2 (Os10g42110) OsBSK3 147

(Os04g58750) and OsBSK4 (Os03g61010) Similar to AtBSKs these OsBSKs were 148

found to contain a N-terminal kinase domain and a C-terminal TPR domain In addition 149

several distinctive features of the kinase domain in AtBSKs were found to be conserved 150

in the OsBSKs including the lack of a classical GXGXXG motif (glycine-rich loop) the 151

presence of an alanine gatekeeper and amino acid substitutions within the conserved 152

HRD and DFG motifs (Figure S1) 153

In preliminary semi-quantitative reverse transcription-PCR (RT-PCR) analyses we found 154

that the expression of OsBSK3 in rice root and leaf tissues was up-regulated by BR 155

treatment (Figure S2) therefore we selected OsBSK3 for further analysis When 156

overexpressed in Arabidopsis OsBSK3 rescued the dwarf phenotypes of the BR 157

synthesis mutant det2 the weak BR signaling mutant bri1-5 and the strong BR signaling 158

mutant bri1-116 (Figure 1AndashD) Compared with bri1-5 control plants expression of the 159

BR synthesis gene CPD was greatly reduced in the transgenic plants overexpressing 160

OsBSK3 (Figure 1E) indicating that overexpression of OsBSK3 activates BR signaling 161

in Arabidopsis 162

Membrane localization is required for the regulation of BR signaling by OsBSK3 163

An analysis by confocal microscopy of transgenic plants overexpressing OsBSK3 with a 164

C-terminal yellow fluorescent protein (OsBSK3-YFP) or green fluorescent protein tag 165


9

(OsBSK3-GFP) showed that OsBSK3 was localized to the plasma membrane in both 166

Arabidopsis and rice seedlings (Figures 2A and S3) Sequence analysis revealed that 167

OsBSK3 does not possess a transmembrane domain however it contains a potential 168

myristoylation site at its N-terminus which serves as a membrane localization signal 169

(Thompson and Okuyama 2000) To examine whether this myristoylation site is critical 170

for the membrane localization and biological function of OsBSK3 we produced 171

transgenic Arabidopsis plants expressing a mutant version of OsBSK3 with a disrupted 172

myristoylation site (the second and third glycine residues were substituted with alanine 173

G2A) and YFP fused to the C-terminus As predicted at an expression level below that of 174

OsBSK3-YFP G2A-YFP was detected throughout the transgenic cells by confocal 175

microscopy (Figure 2A) Consistent with these observations OsBSK3-YFP was detected 176

only in the membrane fraction by immunoblotting while G2A-YFP was mostly detected 177

in the soluble fraction in the transgenic seedlings (Figure 2B) Myristoylation-mediated 178

membrane localization is critical for the biological function of OsBSK3 When 179


10

overexpressed in bri1-5 plants G2A was unable to rescue the semi-dwarf phenotype of 180

the mutants (Figure 2C and D) 181

OsBSK3 is a direct substrate of OsBRI1 182

To test whether OsBSK3 interacts directly with the rice BR receptor OsBRI1 a 183

bimolecular fluorescence complementation (BiFC) assay was performed As shown in 184

Figure 3A tobacco epidermal cells co-expressing OsBSK3 fused to the C-terminal half of 185

cyan fluorescent protein (OsBSK3-cCFP) and full-length OsBRI1 fused to the N-terminal 186

half of YFP (OsBRI1-nYFP) showed strong fluorescence at the plasma membrane 187

whereas tobacco cells co-expressing OsBSK3-cCFP and nYFP showed very weak 188

fluorescence (Figure 3A) The in vivo interaction of OsBRI1 with OsBSK3 was further 189

confirmed using a split luciferase complementation assay A strong luciferase signal was 190

detected in tobacco leaf epidermal cells co-expressing OsBSK3 fused with the C-terminal 191

half of luciferase (OsBSK3-cLuc) and full-length OsBRI1 fused with the N-terminal half 192

of luciferase (OsBRI1-nLuc) but not in cells co-expressing OsBSK3-cLuc and the nLuc 193


11

empty vector or OsBRI1-nLuc and the cLuc empty vector (Figure 3B and C) 194

An in vitro kinase assay showed that OsBSK3 could be phosphorylated by OsBRI1 195

(Figure 3D) To elucidate the effect of phosphorylation by OsBRI1 on the biological 196

function of OsBSK3 lambda phosphatase-treated recombinant OsBSK3 (see the 197

Materials and Methods section) was phosphorylated in vitro by OsBRI1 digested with 198

trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LCMSMS) 199

Mass spectrometry did not identify any phosphopeptides in the lambda 200

phosphatase-treated OsBSK3 protein however Ser213 and Ser215 were found to be 201

phosphorylated in OsBSK3 that had been co-incubated with OsBRI1 suggesting that 202

these residues are specific OsBRI1 phosphorylation sites (Table 1 and Figure S4) To 203

investigate whether these two sites are phosphorylated in vivo OsBSK3 was 204

immunoprecipitated with anti-myc antibodies from 2-week-old rice seedlings 205

overexpressing OsBSK3 with a C-terminal myc tag digested with trypsin and analyzed 206


12

by LCMSMS Our results indicate that the peptide SYSTNLAFTPPEYMR which 207

contained Ser213 and Ser215 was indeed phosphorylated in vivo and that the 208

phosphorylation site was either Ser213 or Ser215 (Table 1 and Figure S5A) 209

Ser215 Phosphorylation is critical for the biological function of OsBSK3 210

To determine the physiological significance of the OsBRI1 phosphorylation sites in 211

OsBSK3 we generated multiple versions of OsBSK3 by site-directed mutagenesis 212

(S213A and S215A) to eliminate phosphorylation at these residues We also substituted 213

Ser213 or Ser215 with glutamate (generating S213E- and S215E-substituted OsBSK3 214

respectively) to mimic constitutive OsBRI1 phosphorylated forms of OsBSK3 These 215

mutant forms of OsBSK3 were overexpressed in bri1-5 plants and examined for their 216

ability to rescue the dwarf phenotype of the mutant Surprisingly replacement of Ser213 217

with either alanine or glutamate had little effect on the ability of OsBSK3 to rescue the 218

dwarf phenotype of the bri1-5 mutant plants (Figure 4A) suggesting that the 219

phosphorylation of Ser213 does not contribute to the regulatory role of OsBSK3 in BR 220

signaling In comparison when the S215A-substituted version of OsBSK3 was expressed 221

at a level similar to that of wild-type OsBSK3 no rescue of the bri1-5 phenotype was 222

observed Furthermore S215A had a dominant-negative effect plants overexpressing the 223

S215A version of OsBSK3 were smaller than the non-transgenic controls when grown in 224

the light under the same conditions and the severity of dwarfism was closely related to 225

the level of expression of the mutant protein (Figure 4B) When overexpressed 226

S215E-substituted OsBSK3 significantly rescued the dwarf phenotype of the bri1-5 227

mutant (Figure 4B) The leaves of bri1-5 mutant plants overexpressing S215E-substituted 228

OsBSK3 were similar in length to those of wild-type (Wassilewskija WS) control plants 229

(Figure S6) Meanwhile the leaves stems and siliques of the S215E 230

mutant-overexpressing plants were curled and twisted (Figure S6) 231

Similarly a peptide containing Ser210 (equivalent to Ser213 of OsBSK3) and Ser212 232


13

(equivalent to Ser215 of OsBSK3) from AtBSK3 was found to be phosphorylated in vivo 233

(Table 1 and Figure S5B) Meanwhile S212A-substituted AtBSK3 lost its ability to 234

rescue the mutant phenotype of bri1-5 plants and the substitution of Ser212 with 235

glutamate enhanced the ability of AtBSK3 to rescue the dwarf phenotype of bri1-5 236

mutant plants compared with wild-type AtBSK3 under overexpression conditions (Figure 237

S7) Taken together these data suggest that the mechanism in which the phosphorylation 238

of BSK3 regulates BR signaling is conserved between rice and Arabidopsis 239

When grown in the dark the etiolated hypocotyls of wild-type and S215E-substituted 240

OsBSK3-expressing plants were significantly longer than those of bri1-5 plants whereas 241

the hypocotyls of S215A-substituted OsBSK3-expressing plants were similar to those of 242

non-transgenic mutant control plants (Figure 4C and D) When grown in the presence of 243

the BR biosynthesis inhibitor propiconazole (PCZ 025 μM) the growth-promoting 244

effect of wild-type OsBSK3 became less distinguishable whereas the etiolated 245

hypocotyls of the S215E-overexpressing bri1-5 mutant plants were wavy twisted and 246

nearly insensitive to PCZ treatment (Figure 4C and D) Similar hypocotyl phenotypes 247

were observed in bzr1-1D a mutant that exhibits constitutively active BR signaling 248

suggesting that the S215E substitution produced constitutively active BR signaling 249

Consistent with these growth phenotypes quantitative RT-PCR (qRT-PCR) showed that 250

the expression levels of CPD were decreased in bri1-5 plants overexpressing wild-type or 251

S215E-substituted OsBSK3 while they were unchanged in bri1-5 plants overexpressing 252

S215A-substituted OsBSK3 (Figure 4E) 253

It was previously reported that the phosphorylation of AtBSK1 by AtBRI1 increases the 254

binding affinity of AtBSK1 for the downstream signaling component AtBSU1 (Kim et al 255

2009) Although a sequence homology search showed that the rice genome contains 256

several AtBSU1 orthologs evidence showing that the BSU1 orthologs in rice regulate BR 257

signaling in a manner similar to AtBSU1 is lacking Therefore we used AtBSU1 to 258

investigate whether the identified OsBRI1 phosphorylation sites in OsBSK3 contribute to 259


14

BSU1 binding Wild-type and several mutated versions of OsBSK3 (S213A S213E 260

Y214F Y214E S215A and S215E) were purified from Escherichia coli using a GST tag 261

separated by SDS-PAGE blotted onto nitrocellulose membranes and incubated with 262

purified MBP-tagged AtBSU1 (MBP-BSU1) the overlay signal was detected using 263

horseradish peroxidase-conjugated anti-MBP antibodies As shown in Figure 4F 264

S215E-substituted OsBSK3 exhibited strong affinity for AtBSU1 whereas no interaction 265

was detected between AtBSU1 and the other forms of OsBSK3 The stronger interaction 266

of S215E-substituted OsBSK3 with AtBSU1 was also confirmed using an in vivo BiFC 267

assay (Figure S8) 268

The TPR domain of OsBSK3 negatively regulates its function 269

Using transgenic plants we discovered that overexpressed OsBSK3 carrying a C-terminal 270

YFP tag rescued the bri1-5 mutant phenotype better than tag-free OsBSK3 (Figure S9) 271

Given that the C-terminus of OsBSK3 contains a TPR domain which is believed to be 272

involved in protein-protein interactions (Allan et al 2011) we tested the function of the 273

TPR domain by overexpressing the kinase domain (amino acids 1ndash390) of OsBSK3 274

(N390) in wild-type plants and in various BR biosynthesis and signaling mutants As 275

shown in Figure S10 overexpressing N390 partially rescued the semi-dwarf phenotype of 276

the bri1-301 mutant while wild-type seedlings overexpressing N390 and OsBSK3 277

showed a bzr1-1D mutant-like axillary branch kink phenotype (Figure S11) caused by 278

BR-regulated organ fusion (Gendron et al 2012) In addition both sets of transgenic 279

plants showed hypersensitivity to epi-BL (eBL)-stimulated hypocotyl elongation and 280

reduced sensitivity to brassinazole (a BR biosynthesis inhibitor BRZ)-inhibited 281

hypocotyl elongation In comparison the observed axillary branch kink phenotype and 282

hypocotyl responses to exogenously applied eBL and BRZ were dramatically enhanced in 283

N390-overexpressing plants suggesting that BR signaling is hyperactivated in 284

Arabidopsis plants overexpressing N390 compared with plants overexpressing full-length 285

OsBSK3 (Figure S11) Consistent with these observations N390 was able to rescue the 286


15

dwarf phenotypes of bri1-5 and bri1-116 better than full-length OsBSK3 in plants 287

expressing the proteins at similar levels (Figure 5A and B) qRT-PCR revealed that CPD 288

expression was greatly reduced in the N390- and OsBSK3-overexpressing bri1-5 mutant 289

plants (Figure 5C) suggesting that the rescue of the mutant phenotype was due to the 290

activation of BR signaling We also overexpressed the TPR domain of OsBSK3 in 291

wild-type Arabidopsis plants and analyzed their response to exogenous eBL As shown 292

overexpression of the TPR domain reduced the sensitivity of the transgenic plants to 293

eBL-stimulated hypocotyl elongation in the light (Figure 5D) 294

The strong ability of N390 to rescue the phenotypes of the BR signaling mutants and the 295

reduced responses to eBL in plants overexpressing the TPR domain suggests that the TPR 296

domain of OsBSK3 serves as an inhibitory domain Thus we next assessed whether the 297

TPR and kinase domains of OsBSK3 (N390) could interact with each other directly by a 298

yeast two-hybrid assay In agreement with previous findings (Sreeramulu et al 2013) 299


16

OsBSK3 was able to interact with itself but the interaction was relatively weak (Figure 300

6A) When OsBSK3 was fragmented the N390 or TPR domain (amino acids 391ndash502) 301

interacted strongly with OsBSK3 Furthermore although neither the N390 nor the TPR 302

domain was able to bind to itself they interacted strongly with each other (Figures 6A 303

and S12A) 304

Besides BRI1 and BSU1 BSK family proteins are able to bind directly with BIN2 305

(Sreeramulu et al 2013) To further investigate the effect of the TPR domain of OsBSK3 306


17

or AtBSK3 on the proteinrsquos physiological function the ability of AtBSU1 AtBIN2 307

full-length OsBSK3 N390 full-length AtBSK3 and the kinase domain of AtBSK3 308

(N334) to interact was examined AtBSU1 did not interact with full-length OsBSK3 or 309

AtBSK3 in a yeast two-hybrid assay whereas a strong interaction between AtBSU1 and 310

N390 or N334 was detected (Figure 6B) In comparison AtBIN2 interacted with both 311

full-length and the kinase domain of OsBSK3 or AtBSK3 These data suggest that the 312

TPR domain prevented both OsBSK3 and AtBSK3 from binding with AtBSU1 but not 313

AtBIN2 Our yeast two-hybrid data were further validated using an in vitro overlay assay 314

in which the kinase domain or full-length version of OsBSK3 (N390 and OsBSK3 315

respectively) or AtBSK3 (N334 and AtBSK3 respectively) carrying a 6His-tag were dot 316

blotted onto a nitrocellulose membrane at a 11 molar ratio and incubated with 317

MBP-AtBSU1 As shown in Figure 6C MBP-AtBSU1 bound more strongly with the 318

kinase domains of AtBSK3 and OsBSK3 than with the full-length versions of the proteins 319

This result was confirmed by a split luciferase assay (Figure S12B) 320

If OsBSK3rsquos TPR domain interacts with its kinase domain to prevent the protein from 321

binding to the downstream target BSU1 could the phosphorylation of OsBSK3 by 322

OsBRI1 prevent its TPR and kinase domains from binding To test this hypothesis a 323

GST-TPR fusion protein was used to pull down N390-6His which was pre-incubated 324

with the OsBRI1 kinase domain in the presence or absence of ATP Our findings indicate 325

that unphosphorylated N390 bound the TPR domain much more strongly than 326

phosphorylated N390 (Figure 6D) Furthermore quantitative yeast two-hybrid and split 327

luciferase assays showed that the binding of the kinase and TPR domains of OsBSK3 was 328

reduced when the S215E-substituted version of the protein was used and increased when 329

the S215A-substituted version of the protein was used (Figures 6E and S12C) 330

OsBSK3 regulates BR signaling in rice 331

To test whether OsBSK3 regulates BR signaling in rice we overexpressed both 332


18

full-length and the kinase domain of OsBSK3 in rice and measured the lamina joint angle 333

and root length which are typically regulated by BR signaling in the transgenic plants 334

Seedlings overexpressing both full-length and the kinase domain of OsBSK3 showed a 335

significantly increased leaf bending angle (Figure 7A and B) However at a similar 336


19

expression level increased leaf bending and root growth inhibition were observed in 337

BL-treated seedlings overexpressing N390 but not from seedlings overexpressing full 338

length OsBSK3 (Figure 7CndashD) We also analyzed the expression levels of the BR 339

biosynthesis genes DWARF and D2 which were previously shown to be 340

feedback-regulated by BR signaling in rice in plants overexpressing N390 or OsBSK3 341

As shown in Figure 7E the expression of DWARF and D2 was reduced in both types of 342

transgenic plants however it was lower in the N390-overexpressing seedlings than in the 343

OsBSK3-overexpressing seedlings Taken together these results suggest that BR 344

signaling was activated in the OsBSK3- and N390-overexpressing rice seedlings Similar 345

to our finding in Arabidopsis the kinase domain of OsBSK3 was more efficient at 346

activating BR signaling than full-length OsBSK3 347

While screening the transgenic rice seedlings we identified several OsBSK3 348

co-suppression (CS) lines qRT-PCR revealed that the expression of endogenous OsBSK3 349

in the CS plants was almost undetectable Furthermore the transcript levels of OsBSK1 350

OsBSK2 and OsBSK4 were all significantly reduced (Figure 7F) The CS seedlings were 351

all smaller in size and had a reduced leaf bending angle (Figure 7F) Similar to a weak 352

OsBRI1 loss-of-function mutant when the plants were full grown the elongation of the 353

second internode was greatly reduced in the CS plants (Figure 7G) Consistent with these 354

phenotypes the expression level of the BR biosynthesis gene OsDWARF was increased in 355

the CS lines (Figure 7H) Moreover when grown in the field the seeds of the 356

N390-overexpressing plants were longer (not wider or thicker) and heavier while the 357

seeds from the CS plants were smaller and lighter than seeds harvested from wild-type 358

plants which were grown alongside the transgenic plants (Figure 7I and J) These data 359

strongly suggest that OsBSK3 expression is critical for the activation of BR signaling in 360

rice 361

We also overexpressed wild-type or S215E-substituted N390 (N390-S215E) in the weak 362

OsBRI1 mutant d61-2 The overexpression of N390 in d61-2 did not alter the mutant 363


20

phenotype of the plants However overexpression of N390-S215E significantly increased 364

the leaf bending angle in young d61-2 mutant plants and the leaves of the full-grown 365

transgenic plants were less erect (Figure 8A and B) The seeds of the 366

N390-S215E-overexpressing plants were much larger than those of the d61-2 control 367

plants (Figure 8C) qRT-PCR revealed that the expression of DWARF and D2 was 368

significantly reduced in the N390-S215E-overexpressing d61-2 mutant plants suggesting 369

that BR signaling was activated (Figure 8D) Taken together our results suggest that 370

similar to our observation in Arabidopsis the ability of the various forms of OsBSK3 to 371

activate BR signaling in rice was as follows N390-S215E gt N390 gt full-length OsBSK3 372

373


21

DISCUSSION 374

As major growth-promoting hormones BRs play important roles in regulating 375

yield-related traits in rice plants including leaf angle tillering plant height and grain 376

filling (Hong et al 2004 Vriet et al 2012) Compared with the elaborate BR signaling 377

mechanism discovered in Arabidopsis BR signaling in rice is not well understood 378

However the severe growth-inhibiting phenotypes caused by the mutation of BRI1 379

orthologs in rice (Nakamura et al 2006) tomato (Koka et al 2000) pea (Nomura et al 380

2003) and barley (Gruszka et al 2011) suggest that the BRI1-mediated BR signaling 381

pathway is conserved across the plant kingdom In addition to OsBRI1 orthologs of other 382

Arabidopsis BR signaling components such as OsBAK1 (Li et al 2009) OsGSK2 (Tong 383

et al 2012) and OsBZR1 (Bai et al 2007) have been found to regulate BR signaling in 384

rice however the molecular mechanisms leading from the activation of OsBRI1 by BRs 385

to the regulation of gene expression are mostly unknown In this study we identified four 386

BSK orthologs in the rice genome by a sequence homology search Overexpression of the 387

kinase domain of OsBSK3 in rice plants reduced the expression of the BR biosynthesis 388

genes DWARF and D2 and increased the sensitivity of the transgenic seedlings to 389

eBL-induced leaf bending and eBL-inhibited root elongation Consistent with our 390

overexpression data simultaneous knockdown of the four OsBSKs reduced the leaf 391

bending angle and increased DWARF expression in rice Taken together our results 392

suggest that OsBSK3 positively regulates BR signaling in rice 393

Despite the fact that OsBSK3 does not contain a transmembrane domain subcellular 394

localization studies showed that it is localized to the plasma membrane by an N-terminal 395

myristoylation site This localization pattern is critical for the activation of BR signaling 396

by OsBSK3 the mutation of this myristoylation site not only changed the localization of 397

OsBSK3 from the plasma membrane to the cytoplasm it also impaired the ability of 398

OsBSK3 to rescue the mutant phenotype of bri1-5 Similar observations have been 399

reported for other RLCKs including AtBSK1 (Shi et al 2013) AtLIP1 AtLIP2 (Liu et 400


22

al 2013) CST (Burr et al 2011) and TPK1b (AbuQamar et al 2008) indicating that 401

lipidation-mediated membrane localization is a general feature of these 402

membrane-associated RLCKs Correlated with its plasma membrane localization pattern 403

BiFC and split luciferase assays showed that OsBSK3 interacted directly with and was 404

phosphorylated by the membrane-localized BR receptor OsBRI1 Together these results 405

suggest that the role of OsBSK3 in regulating BR signaling is similar to that of AtBSKs 406

which transduce BR signals from BRI1 to downstream signaling component BSU1 This 407

hypothesis is further supported by the observation that S215E-substituted OsBSK3 408

which mimicked the constitutively phosphorylated form of OsBSK3 bound BSU1 more 409

strongly than wild-type OsBSK3 410

Even though phosphorylation by AtBRI1 increases AtBSK1 binding to the downstream 411

phosphatase BSU1 (Kim et al 2009) direct evidence showing that the phosphorylation 412

of BSKs by BRI1 activates BR signaling in vivo is lacking By mass spectrometry we 413

identified Ser213 and Ser215 of OsBSK3 as OsBRI1 phosphorylation sites Site-directed 414

mutagenesis revealed that inhibiting the phosphorylation of OsBSK3 at Ser215 by OsBRI1 415

prevented OsBSK3 from rescuing the mutant phenotype of bri1-5 plants while 416

substituting Ser215 with glutamate not only rescued the bri1-5 mutant phenotype it also 417

made the transgenic plants insensitive to 025 μM PCZ-inhibited hypocotyl elongation 418

Similarly the phosphorylation of AtBSK3 at Ser212 (equivalent to Ser215 of OsBSK3) 419

activated BR signaling in Arabidopsis This conservative serine residue was previously 420

shown to be a major AtBRI1 phosphorylation site in AtBSK1 (Ser230) and AtCDG1 421

(Ser234) it is also important for the activation of AtBSU1 by AtBSK1 and AtCDG1 (Kim 422

et al 2009 2011) Together these results suggest that the mechanism by which BRI1 423

regulates BR signaling through the phosphorylation of BSKs is conserved in both 424

Arabidopsis and rice Although this idea was mostly tested using transgenic Arabidopsis 425

plants the partial rescue of the d61-2 mutant phenotype by overexpression of the 426

S215E-substituted form of the OsBSK3 kinase domain (but not the wild-type form) 427


23

suggests that a similar mechanism functions in rice plants 428

With 72 homology to AtBSK3 OsBSK3 contains all of the key features of AtBSKs A 429

protein structure analysis of AtBSK8 suggested that AtBSKs are constitutively inactive 430

protein kinases (Grutter et al 2013) However it was reported that AtBSK1 might 431

contain weak Mn2+-dependent kinase activity (Shi et al 2013) We also detected a weak 432

OsBSK3 autophosphorylation signal during our in vitro kinase assay The 433

autoradiographic signal was stronger in the presence of Mn2+ and it was increased by 434

OsBRI1 phosphorylation (Figure S13) However the signal was so weak that we had to 435

expose the dried gel under a storage phosphor screen for 1 week in order to obtain a clear 436

image Therefore we cannot confidently claim that OsBSK3 is an active kinase based on 437

this result To further investigate whether the kinase activity of OsBSK3 is an essential 438

part of its BR signaling function we mutated two invariable amino acids that are 439

considered to be critical for the kinase activity of OsBSK3 to create two mutant forms 440

(K89E and K89E D183A) of the kinase by site-directed mutagenesis (Grutter et al 2013) 441

Surprisingly when overexpressed these ldquokinase-deadrdquo forms of OsBSK3 were unable to 442

rescue the semi-dwarf phenotype of bri1-5 mutant plants (Figure S14) suggesting that 443

the kinase activity of OsBSK3 is required for its ability to transduce BR signals As a 444

binding partner-dependent activation mechanism has been shown to regulate AtRRK1 445

family RLCKs (Dorjgotov et al 2009) whether a similar mechanism exists for BSK 446

family proteins should be examined in a future study 447

Besides BSKs AtCDG1 family RLCKs positively regulate BR signaling (Kim et al 448

2011) Similar to AtBSKs AtCDG1 can interact directly with AtBRI1 and activate the 449

downstream component AtBSU1 upon BRI1 phosphorylation However the 450

overexpression of AtCDG1 in an AtBSK3 T-DNA insertion mutant could not rescue its 451

BR-insensitive phenotype suggesting that AtCDG1 regulates BR signaling differently 452

from AtBSK3 (Kim et al 2011) Here we found that plants overexpressing 453

S215E-substituted OsBSK3 had cdg1-D-like curled and twisted stems leaves and 454


24

siliques (Muto et al 2004) As cdg1-D is a gain-of-function mutant in which CDG1 is 455

overexpressed due to the insertion of four 35S enhancers into its promoter sequence the 456

similar phenotypes of the CDG1- and S215E-overexpressing plants suggest that OsBSK3 457

and CDG1 regulate plant development via similar mechanisms 458

A common feature of BSK family RLCKs is the presence of an N-terminal kinase domain 459

and a C-terminal TPR domain however the role of the TPR domain in regulating the 460

biological function of BSKs is unknown Although it is generally accepted that the TPR 461

domain acts mostly as a scaffold to promote the formation of multi-protein complexes 462

(Allan et al 2011) our study shows that the TPR domain of both AtBSK3 and OsBSK3 463

acts as an autoinhibitory domain that binds to the kinase domain of BSK3 and prevents it 464

from binding to and activating BSU1 Our in vitro pull down and quantitative yeast 465

two-hybrid assay results suggest that when OsBSK3 was phosphorylated by OsBRI1 the 466

binding affinity of its TPR domain for its kinase domain was greatly reduced A protein 467

overlay assay showed that phosphorylation at Ser215 greatly increased the binding of 468

OsBSK3 to BSU1 Taken together our results suggest that the binding of OsBSK3 with 469

AtBSU1 is regulated by BRI1-dependent phosphorylation 470

Based on our results we propose a working model for BSKs in which the TPR domain 471

binds with the kinase domain to prevent BSKs from binding to and activating BSU1 472

when BR signaling is turned off The phosphorylation of BSKs by BR-activated BRI1 473

frees the kinase domain of BSKs from the TPR domain and increases the binding affinity 474

of BSKs for BSU1 promoting the dephosphorylation of BIN2 by BSU1 via a still 475

unknown mechanism 476

477

MATERIALS AND METHODS 478

Plant materials and growth 479


25

The Arabidopsis bri1-5 mutant is of the Wassilewskija (WS) ecotype while the det2 and 480

bri1-116 mutants are of the Columbia (Col) ecotype For the observation of growth 481

phenotypes Arabidopsis seedlings were grown in a greenhouse at 22 degC under long-day 482

(LD) conditions (16 h of light8 h of dark) at a light intensity around 100 μmolmiddotm-2middots-1 483

For the hypocotyl growth assays Arabidopsis seedlings were grown on agar medium 484

containing half-strength Murashige and Skoog (12 MS) salts 1 sucrose and the 485

indicated concentration of eBL in the light or the BR biosynthesis inhibitor PCZ or BRZ 486

in the dark at 22 degC for 1 week before taking pictures for measurement using ImageJ The 487

average ratio and standard deviation were calculated based on values collected from at 488

least three biological repeats with at least 15 plants per experiment The experiments 489

included in this study were performed at least three times with similar results 490

Representative data from one repetition are shown in the figures 491

Oryza sativa ssp japonica cultivar Dongjin was used for all transgenic experiments in 492

rice OsBRI1 mutant d61-2 is of the Taichung 65 background For the BR-induced lamina 493

joint bending assay rice seeds were germinated in double-distilled water at 28 degC in the 494

dark for 4 days The seedlings were then transferred to soil and grown for 10 additional 495

days under the same conditions before treatment with different concentrations of eBL at 496

the leaf lamina joint to stimulate bending The seedlings were allowed to grow 3 497

additional days before taking photos the leaf bending angle for each seedling was 498

calculated using ImageJ software The average ratio and standard deviation were 499

calculated based on values collected from at least three biological repeats with 10ndash15 500

plants per experiment 501

Overexpression of OsBSK3 in Arabidopsis and rice 502

Full-length wild-type and mutated OsBSK3 or amino acids 1ndash390 of the wild-type 503

OsBSK3 coding sequence (N390) without the stop codon were cloned into the binary 504

vectors pEarlygate 101 (p101) pEarlygate 100 (p100) PMDC83 or pCAMBIA-1390 505


26

and then introduced into Agrobacterium tumefaciens strain GV3101 or EHA105 The 506

constructs were transformed into Arabidopsis by the GV3101-mediated floral dip method 507

Multiple independent T3 homozygous transgenic lines were used for the phenotype 508

analysis shown in this study the reported results were consistent in these independent 509

lines Transgenic rice plants were generated by EHA105-mediated callus transformation 510

(Yang et al 2004) T2 homozygous transgenic rice seedlings were used for the 511

phenotype analysis unless indicated otherwise 512

Gene expression analysis by quantitative real-time RT-PCR 513

Total RNA was extracted using TRIzol reagent (Invitrogen Carlsbad CA) First-strand 514

cDNA was synthesized using M-MLV Reverse Transcriptase (Takara Bio Inc Otsu 515

Japan) according to the manufacturerrsquos instructions A SYBR Premix Ex TaqTM (Tli 516

RNase H Plus) kit (Takara Bio Inc) was used for the real-time quantitative PCR analysis 517

of gene expression with an ABI PRISM 7500 Real-Time System The primer pairs used 518

were 5-ttgctcaactcaaggaagag-3 and 5-tgatgttagccactcgtagc-3 for CPD (At5g05690) 519

5-caaatccaaaaccctagaaaccgaa-3 and 5-atctcccgtaggacctgcactg-3 for UBC30 520

(At5g56150) 5-agctgcctggcactaggctctacagatcac-3 and 5- 521

atgttgtcggagatgagctcgtcggtgagc-3 for D2 (Os01g10040) 5-caagcagggacccagtttcat-3 and 522

5-ccaggatgtccagcatcgact-3 for DWARF (Os03g40540) 5rsquo-acacctccagagtatttgagaaatg-3rsquo 523

and 5rsquo-aactagaatctgatggattgctgtc-3rsquo for OsBSK1 (Os03g04050) 5rsquo-caccctttccaagcatctct-3rsquo 524

and 5rsquo-tataacttttcccatcgcgg-3rsquo for OsBSK2 (Os10g42110) 525

5rsquo-cgcggatcccaccgcaaagcctgaggtggccag-3rsquo and 5rsquo-caccatgggcgggcgcgtgtccaag-3rsquo for 526

OsBSK3 (Os04g58750) 5rsquo-actgggagacaaagccattgag-3rsquo and 5rsquo-gccttctaagcaagagtccatca-3rsquo 527

for OsBSK4 (Os03g61010) 5-agcaactgggatgatatgga-3 and 5-cagggcgatgtaggaaagc-3 528

for OsACTIN1 (Os03g50885) The expression level of CPD was normalized against that 529

of UBC30 in Arabidopsis while the expression levels of DWARF and D2 were 530

normalized against that of OsACTIN1 in rice The relative gene expression level is 531

presented as a ratio to the expression level in the control sample The average ratio and 532


27

standard deviation were calculated based on values collected from at least three 533

biological repeats 534

Fractionation of membrane proteins 535

Microsomal protein was prepared according to Tang et al (2012) with slight 536

modifications In brief Arabidopsis seedlings were ground in buffer H (25 mM HEPES 537

10 glycerol 033 M sucrose 06 polyvinylpyrrolidone 5 mM ascorbic acid 5 mM 538

EDTA 25 mM NaF 1 mM sodium molybdate 2 mM imidazole 1 mM activated sodium 539

vanadate 5 mM DTT 1 microM E-64 1 microM bestatin 1 microM pepstatin 2 microM leupeptin and 1 540

mM PMSF pH 75) at 4 degC The homogenate was filtered through two layers of 541

Miracloth and centrifuged at 10000 x g for 15 min to pellet cell debris and organelles 542

The supernatant was then spun at 60000 x g for 60 min to pellet microsomes The 543

supernatant (soluble proteins) and microsome pellet (membrane proteins) were boiled in 544

2times SDS extraction buffer (125 mM Tris-HCl pH 68 2 β-mercaptoethanol 4 SDS 545

20 glycerol and 025 bromophenol blue) for 5 min separated by 10 SDS-PAGE 546

transferred to a nitrocellulose membrane (EMD Millipore Billerica MA) and detected 547

with anti-GFP antibodies 548

In vivo protein-protein interaction assays 549

Yeast two-hybrid and BiFC assays were performed according to Gampala et al (2007) 550

The full-length coding sequences of OsBSK3 and OsBRI1 (Os01g52050) were cloned 551

in-frame into the BiFC expression vectors pX-NYFP and pX-CCFP The vectors were 552

then transformed into A tumefaciens strain GV3101 and co-infiltrated into 4-week-old 553

tobacco leaves At 36ndash48 h after infiltration YFP fluorescence was observed by confocal 554

microscopy (Zeiss LSM 510 Jena Germany) 555

For the split luciferase assay the full-length coding sequence of OsBRI1 was cloned into 556

pCAMBIA-NLuc (Chen et al 2008) with the N-terminal luciferase sequence fused to 557

the C-terminus of OsBRI1 The C-terminal luciferase sequence was amplified by PCR 558


28

from pCAMBIA-CLuc (Chen et al 2008) and added to the C-terminus of the full-length 559

OsBSK3 coding sequence in pENTRSDD-TOPO (Invitrogen) followed by sub-cloning 560

into pEarlygate 100 using LR Clonase (Invitrogen) The resulting OsBRI1-NLuc- and 561

OsBSK3-CLuc-expressing vectors were introduced to A tumefaciens strain GV3101 and 562

infiltrated into Nicotiana benthamiana leaves as described previously (Gampala et al 563

2007) At 36ndash48 h after infiltration the tobacco leaves were sprayed with 25 mgml 564

luciferin (Gold Biotechnology Inc Olivette MO) and kept in the dark for 6 min to 565

quench chloroplast fluorescence Images showing luciferase bioluminescence were taken 566

using a low-light cooled CCD imaging apparatus (Andor Technology Belfast UK) with 567

the temperature of the camera pre-cooled to -96 degC (exposure time 500 s 1times1 binning) 568

Relative firefly luciferase activity was detected using a Centro LB 960 Microplate 569

Luminometer (Berthold Technologies Bad Wildbad Germany) At 36ndash48 h after 570

infiltration discs were punched from the tobacco leaves (03 cm in diameter) and placed 571

into 96-well plates The leaf discs were kept in the dark for 6 min to quench the 572

fluorescence signal 50 μl of 25 mgml luciferin (Gold Biotechnology Inc) were then 573

injected and the bioluminescence signal was recorded immediately for 10 s The average 574

ratio and standard deviation were calculated based on values collected from at least three 575

biological repeats with 5ndash6 independent measurements per experiment 576

Site-directed mutagenesis 577

The full-length coding sequence of OsBSK3 (without the stop codon) was cloned into 578

pENTRSDD-TOPO (Invitrogen) and used as the template for all site-directed 579

mutagenesis experiments Site-directed mutagenesis was achieved using PCR which was 580

performed with 18 cycles in a 10-μl reaction mixture The PCR product was digested 581

overnight using DpnI (Takara Bio Inc) and 1 μl of the digested product was used to 582

transform E coli strain DH5α Following the verification of each mutation by sequencing 583

the mutated forms of OsBSK3 were incorporated into a gateway-compatible destination 584


29

vector (pEarlygate 101 pGEX4T-1 gc-pGBKT7 or gc-pCAMBIA-1390) using LR 585

Clonase (Invitrogen) 586

Protein purification and in vitro protein-protein interaction assays 587

GST- MBP- and 6His-tagged recombinant proteins were expressed in E coli and 588

purified by standard protocols using glutathione Sepharose 4B beads (GE Healthcare 589

Little Chalfont UK) amylose agarose beads (New England Biolabs Ipswich MA) or 590

Ni-NTA resin (Qiagen Hilden Germany) The purified recombinant proteins were 591

concentrated by ultrafiltration using Centricon centrifuge tubes (EMD Millipore) with a 592

10-kDa molecular weight cut-off 593

For the dot blot assays around 1 μmol each of recombinant 6His-OsBSK3 and 594

6His-N390 was dot blotted onto a nitrocellulose membrane The membrane was allowed 595

to air dry for 15 min in a cold room and then incubated in PBS containing 5 nonfat milk 596

Following incubation with 1 μgml MBP-AtBSU1 followed by peroxidase-labelled 597

anti-MBP antibodies the overlay signal was detected using SuperSignal West Dura 598

Chemiluminescence Reagent (Pierce Rockford IL) For the in vitro pull down assays 2 599

μg of 6His-N390 were incubated overnight with 1 μg of MBP-tagged kinase domain of 600

OsBRI1 in the presence or absence of 01 mM ATP Glutathione Sepharose 4B beads 601

bound GST-TPR were added to the reaction and the mixture was rotated at 4 degC for 2 h 602

The beads were then washed three times with PBS and the proteins were eluted by 603

boiling in 2times SDS sample buffer for 5 min After SDS-PAGE the proteins were 604

transferred to a nitrocellulose membrane and the pull down signal was detected using 605

anti-6His or -GST antibodies 606

In vitro kinase assays and identification of the phosphorylation sites by mass 607

spectrometry 608

In vitro phosphorylation assays were performed in a mixture containing 25 mM Tris-HCl 609

pH 75 10 mM MgCl2 or 10 mM MnCl2 100 mM NaCl 1 mM DTT 100 μM ATP 5-10 610


30

μCi of 32P-γATP 05ndash1 μg of GST-OsBRI1KD and 2ndash5 μg of GST-OsBSK3 The 611

mixtures were incubated at 30 degC for 3 h with constant shaking The reactions were 612

stopped by the addition of 6times SDS sample buffer and incubation at 65 degC for 10 min 613

Protein phosphorylation was analyzed by SDS-PAGE and autoradiography 614

To identify the OsBRI1 phosphorylation sites on OsBSK3 GST-OsBSK3 attached to 615

glutathione sepharose 4B beads was first incubated with lambda phosphatase for 6 h to 616

generate hypo-phosphorylated OsBSK3 because it was reported that purified recombinant 617

proteins can be phosphorylated by anonymous bacterial kinases when expressed in E coli 618

cells or during the protein purification process (Wu et al 2012) After incubation the 619

sepharose beads were washed extensively to remove the lambda phosphatase and eluted 620

with 10 mM glutathione Next 25 μg of lambda phosphatase-treated GST-OsBSK3 were 621

incubated with 5 μg of GST-OsBRI1KD overnight and then separated by SDS-PAGE 622

The Coomassie blue-stained gel containing GST-OsBSK3 was cut into 1ndash2 mm pieces 623

and in-gel digested The extracted peptides were freeze-dried using a SpeedVac 624

resuspended in 01 formic acid (FA) and separated by reverse phase liquid 625

chromatography (LC) with an Easy-Spray PepMapreg column (75 microm x 15 cm Thermo 626

Fisher Scientific Waltham MA) using a nanoACQUITY Ultra Performance LC system 627

(Waters Corp Milford MA) LC was conducted using buffer A (2 acetonitrile and 01 628

FA) and buffer B (98 acetonitrile and 01 FA) A linear gradient from 2ndash30 B 629

followed by washing with 50 B at a flow rate of 300 nlmin was used for peptide 630

separation MSMS was conducted with an LTQ Orbitrap Velos Mass Spectrometer 631

(Thermo Fisher Scientific) using the top six data-dependent acquisition method The 632

sequence included one survey scan in FT mode in the Orbitrap with a mass resolution of 633

30000 followed by six CID scans in LTQ focusing on the first six most intense peptide ion 634

signals whose mz values were not on the dynamically updated exclusion list and whose 635

intensities exceeded a threshold of 1000 counts The CID-normalized collision energy 636

was set to 30 The analytical peak lists were generated from the raw data using PAVA 637


31

software (Guan et al 2011) The MSMS data were searched against the proteome 638

sequences for rice and Arabidopsis from Swiss-Prot using the Protein Prospector search 639

engine (httpprospectorucsfeduprospectormshomehtm) The mass tolerance for 640

precursor ions and fragment ions was set to 20 ppm and 07 Da respectively The 641

maximum number of missed cleavages was set at 2 Serine threonine and tyrosine 642

phosphorylation cysteine carbamidomethylation and methionine oxidation were 643

included in the search as variable modifications 644

645 646


32

Supplemental Data 647

The following materials are available in the online version of this article 648

Supplemental Figure 1 Four BSK orthologs are present in the rice genome 649

Supplemental Figure 2 Semi-quantitative RT-PCR analysis of the expression of the 650 OsBSKs in rice seedlings 651

Supplemental Figure 3 Subcellular localization of OsBSK3 in rice root 652

Supplemental Figure 4 The in vitro OsBRI1-phosphorylated peptides identified by 653 mass spectrometry from OsBSK3 654

Supplemental Figure 5 In vivo-phosphorylated peptides identified by mass 655 spectrometry from immunoprecipitated OsBSK3 or AtBSK3 656

Supplemental Figure 6 Phenotypes of S215E-overexpressing bri1-5 mutant lines 657

Supplemental Figure 7 The phosphorylation of AtBSK3 at Ser212 activates BR 658 signaling in Arabidopsis 659

Supplemental Figure 8 BiFC assays showed that AtBSU1 interacted more strongly 660 with the S215E form of OsBSK3 661

Supplemental Figure 9 OsBSK3 with YFP fused to its C-terminus was more efficient 662 at suppressing the dwarf phenotype of bri1-5 mutant plants 663

Supplemental Figure 10 Overexpression of the kinase domain of OsBSK3 partially 664 suppressed the semi-dwarf phenotype of bri1-301 mutant plants 665

Supplemental Figure 11 BR signaling was hyperactivated in N390-overexpressing 666 Arabidopsis plants 667

Supplemental Figure 12 Quantitative analysis of the interaction between the kinase 668 and TPR domains of OsBSK3 and AtBSU1 669

Supplemental Figure 13 Assay for OsBSK3 autophosphorylation 670

Supplemental Figure 14 Overexpression of the K89E or K89E D183A forms of 671 OsBSK3 could not rescue bri1-5 mutant plants 672

673


33

Table 1 The phosphorylated peptides identified from OsBSK3 and AtBSK3 by 674 LCMSMS 675 Peptidea Measured

[M+H]+ Calculated

[M+H]+ Score P-value Identified

sites Identified in vitro phosphopeptides from OsBSK3 by CID DGKpSYSTNLAFTPPEYMR 21729446 21729308 227 67E-06 S213 DGKSYpSTNLAFTPPEYMR 21729389 21729308 348 21E-09 S215 Identified in vivo phosphopeptides from OsBSK3 by HCD pSYpSTNLAFTPPEYMR 18567945 18567925 48 20E-11 S213 or S215 Identified in vivo phosphopeptides from AtBSK3 by CID pSYpSTNLAFTPPEYLR 18388317 18388361 242 66E-06 S210 or S212 aMethionine sulfoxide is denoted as M phosphoseryl residues are denoted as pS 676 677 678


34

Acknowledgement 679

We thank professor Tomoaki Sakamoto (Nagoya University) for kind providing the d61-2 680 rice mutant 681

682


35

FIGURE LEGENDS 683

Figure 1 OsBSK3 overexpression rescued the mutant phenotypes of det2 bri1-5 and 684

bri1-116 plants A C and D Phenotype of light-grown 4-week-old det2 (A) 7-week-old 685

bri1-5 (C) and 8-week-old bri1-116 (D) mutant plants overexpressing AtBSK3 or 686

OsBSK3 with a C-terminal YFP tag B Expression levels of OsBSK3-YFP and 687

AtBSK3-YFP in the transgenic plants shown in (A) Ponceau S staining of the rubisco 688

large subunit was used as an equal loading control E Quantitative real-time RT-PCR 689

analysis of the expression of CPD in one-week-old wild-type (WS) bri1-5 and 690

OsBSK3-YFP-overexpressing bri1-5 (3B10) plants A one-way ANOVA was performed 691

statistically significant differences are indicated by different lowercase letters (Plt005) 692

693

Figure 2 Membrane localization is crucial for the regulation of BR signaling in 694

Arabidopsis by OsBSK3 A The upper panel shows the G2A mutation Red letters show 695

the mutated amino acid The middle and lower panels show the YFP signal detected by 696

confocal microscopy in one-week-old light-grown OsBSK3-YFP- and 697

G2A-YFP-overexpressing bri1-5 leaves B 100 microg of soluble (S) or microsomal (M) 698

proteins from OsBSK3-YFP- and G2A-YFP-overexpressing bri1-5 mutant plants were 699

separated by SDS-PAGE and immunoblotted using anti-YFP antibodies Coomassie 700

brilliant blue (CBB) staining of the rubisco large subunit was used as an equal loading 701

control C Seven-week-old bri1-5 mutant plants overexpressing OsBSK3-YFP or 702

G2A-YFP G2A-1 and -8 are two independent transgenic lines D OsBSK3-YFP and 703

G2A-YFP expression in the 3-week-old transgenic plants shown in (C) Ponceau S 704

staining of the rubisco large subunit was used as an equal loading control 705

706

Figure 3 OsBSK3 is a direct substrate of OsBRI1 A and B BiFC (A) and firefly 707

luciferase complementation assays (B) revealed the direct interaction of OsBSK3 with 708

full-length OsBRI1 in 4-week-old N benthamiana leaf epidermal cells The leaves were 709


36

co-infiltrated with Agrobacterium cells containing the indicated vector pairs Images were 710

collected 36ndash48 h after infiltration DIC differential interference contrast microscopy C 711

Quantitation of the complemented luciferase activity in N benthamiana leaves 712

co-transformed with the indicated vector pairs The experiment was repeated at least three 713

times with consistent results representative results are shown A one-way ANOVA was 714

performed statistically significant differences are indicated by different lowercase letters 715

(Plt005) D An in vitro assay for the phosphorylation of full-length OsBSK3 by OsBRI1 716

717

Figure 4 Functional analysis of the OsBSK3 phosphorylation sites in Arabidopsis 718

A and B Eight-week-old wild type (WS) bri1-5 mutant and bri1-5 mutant 719

overexpressing wild type or a mutated form (S213A S213E S215A and S215E) of 720

OsBSK3 Immunoblotting for the expression of various OsBSK3 forms was conducted 721

using anti-GFP antibodies since the OsBSK3s were produced with a C-terminal YFP tag 722

Ponceau S staining for the rubisco large subunit was used as an equal loading control C 723

The S215E transgenic plants were insensitive to PCZ-inhibited hypocotyl elongation 724

Young seedlings of wild type (WS) bri1-5 or bri1-5 overexpressing a mutated form of 725

OsBSK3 were grown in the dark for 5 days on regular medium or medium containing 726

025 μM PCZ D A quantitative analysis of the hypocotyls of the seedlings shown in (C) 727

Each value in the graph represents the average of at least 20 individual seedlings Error 728

bars represent the standard error (SE) E Quantitative real-time RT-PCR analysis of the 729

CPD expression levels in the seedlings shown in (B) Error bars represent plusmn standard 730

deviation (SD) G A gel overlay analysis of the interaction between AtBSU1 and the 731

various OsBSK3 mutant proteins GST-tagged OsBSK3 proteins were immunoblotted 732

onto a nitrocellulose membrane and probed with MBP-AtBSU1 and horseradish 733

peroxidase-labeled anti-MBP antibodies Total protein was visualized by Ponceau S 734

staining A one-way ANOVA was performed in (D) and (E) statistically significant 735

differences are indicated by different lowercase letters (Plt005) 736


37

737

Figure 5 The TPR domain of OsBSK3 negatively regulates OsBSK3 function A and 738

B Seven- to eight-week-old bri1-5 (A) and bri1-116 mutant (B) plants overexpressing 739

full-length OsBSK3 or the kinase domain of OsBSK3 (N390) The upper panels in (A) 740

and (B) show the expression levels of OsBSK3 and N390 in the transgenic plants C An 741

analysis of the CPD expression level in the 2-week-old seedlings shown in (A) by 742

qRT-PCR D Overexpression of the TPR domain of OsBSK3 reduced the BR sensitivity 743

of the transgenic Arabidopsis plants Plants were grown in 12 MS medium with or 744

without the indicated concentration of eBL at 22 degC under LD conditions for 1 week 745

Representative results from three independent experiments are shown A one-way 746

ANOVA was performed in (C) and (D) Statistically significant differences are indicated 747

by different lowercase letters (Plt005) 748

749

Figure 6 The TPR domain of BSK3 prevents it from interacting with AtBSU1 A 750

Yeast two-hybrid assays of the interaction between full-length OsBSK3 the kinase 751

domain of OsBSK3 (N390) and the TPR domain of OsBSK3 (TPR) B Yeast two-hybrid 752

assays of the interaction between full-length AtBSK3 full-length OsBSK3 the kinase 753

domain of AtBSK3 (N334) and N390 with AtBSU1 AtBIN2 and the TPR domain from 754

OsBSK3 C AtBSU1 showed increased binding with the kinase domains of OsBSK3 and 755

AtBSK3 The recombinant 6His-tagged kinase domain and full-length versions of 756

OsBSK3 (N390 and OsBSK3) or AtBSK3 (N334 and AtBSK3) were dot blotted onto 757

nitrocellulose membranes and then incubated with MBP-AtBSU1 and horseradish 758


staining D OsBRI1 phosphorylation prevents the TPR and kinase domains of OsBSK3 760

from binding GST-TPR on glutathione Sepharose 4B beads was used to pull down 761

6His-tagged N390 which had been incubated with MBP-tagged OsBRI1 kinase domain 762

in the presence (pN390) or absence (N390) of ATP The proteins were blotted onto 763


38

nitrocellulose membranes and detected using anti-His or -GST antibodies E Ser215 764

phosphorylation reduced the binding of the kinase and TPR domains of OsBSK3 Shown 765

are quantitative β-glycosidase activity assays of the interaction between the TPR domain 766

and wild type or Ser215-substituted mutant form of N390 A one-way ANOVA was 767

performed Statistically significant differences are indicated by different lowercase letters 768

(Plt005) 769

770

Figure 7 OsBSK3 regulates the BR response in rice A The lamina joint angle of the 771

second leaves from 2-week-old rice seedlings overexpressing OsBSK3-myc 33 and 38 772

are two independent transgenic lines B Overexpression of the kinase domain of 773

OsBSK3 (N390) enhanced the bending of the lamina joint in the transgenic rice seedlings 774

The upper panel shows the lamina joints of the second leaves from 2-week-old seedlings 775

overexpressing C-terminal myc tag-fused OsBSK3 (BSK3) or kinase domain of OsBSK3 776

(N390) The lower panel shows the expression levels of OsBSK3-myc and N390-myc in 777

the rice seedlings shown above using anti-myc antibodies C Quantitative analysis of 778

BR-stimulated leaf lamina joint bending in OsBSK3- and N390-overexpressing rice 779

seedlings A total of 10 μl of the indicated concentration of eBL was applied to the lamina 780

joint region of 10-day-old light-grown rice seedlings The leaf bending angle was 781

measured 3 days after eBL application D Quantitative analysis of BR-inhibited root 782

elongation in OsBSK3- and N390-overexpressing rice seedlings Seedlings were grown 783

in Hoagland medium with or without 1 μM eBL for 1 week Relative growth was 784

calculated by dividing the root length in eBL by the root length under control conditions 785

E Quantitative real-time RT-PCR analysis of DWARF and D2 expression in 2-week-old 786

rice seedlings overexpressing OsBSK3 or N390 F Left quantitative RT-PCR analysis of 787

the expression of OsBSKs in 2-week-old WT and OsBSK3 CS transgenic rice seedlings 788

Right Phenotypes of the seedlings used in the RT-PCR analysis G Internode length of 789

fully grown WT and CS plants H Quantitative real-time RT-PCR analysis of DWARF 790


39

expression in 2-week-old CS seedlings I Seeds from N390-overexpressing and CS 791

transgenic rice plants J Weights of the seeds shown in (I) A one-way ANOVA was 792

performed in all experiments Statistically significant differences are indicated by 793

different lowercase letters (Plt005) 794

795

Figure 8 Partial rescue of the d61-2 mutant phenotype via overexpression of the 796

S215E-substituted kinase domain of OsBSK3 A The upper panel shows 5-week-old 797

d61-2 mutant plants or d61-2 plants overexpressing C-terminal myc tagged 798

S215E-substituted kinase domain of OsBSK3 (N390-S215E) 201-2 and 28-5 are two 799

independent transgenic lines Arrow exaggerated lamina joint bending in the transgenic 800

seedlings The lower panel shows the expression levels of N390-S215E protein in the two 801

independent transgenic lines shown in the upper panel B Full-grown d61-2 mutant and 802

transgenic d61-2 plants overexpressing N390-S215E (28-5) C Seeds of d61-2 mutant 803

plants and d61-2 mutant plants overexpressing N390-S215E grown in the field D The 804

expression levels of DWARF and D2 in 1-month-old d61-2 mutant plants and d61-2 805

plants overexpressing N390-S215E (28-5) Error bars represent plusmn SE Statistically 806

significant differences are indicated by asterisks (Plt005) 807

808


40

REFERENCES 809 AbuQamar S Chai MF Luo H Song F and Mengiste T (2008) Tomato protein kinase 810

1b mediates signaling of plant responses to necrotrophic fungi and insect herbivory 811 Plant Cell 201964-1983 812

Allan RK and Ratajczak T (2011) Versatile TPR domains accommodate different modes 813 of target protein recognition and function Cell Stress and Chaperones 16353-367 814

Bai MY Zhang LY Gampala SS Zhu SW Song WY Chong K and Wang ZY (2007) 815 Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice Proc 816 Natl Acad Sci USA 10413839-13844 817

Burr CA Leslie ME Orlowski SK Chen I Wright CE Daniels MJ And Liljegren SJ 818 (2011) CAST AWAY a membrane associated receptor-like kinase inhibits organ 819 abscission in Arabidopsis Plant Physiology 156 1837-1850 820

Castelles E and Casacuberta JM (2007) Signalling through kinase defective domains the 821 prevalence of atypical receptor-like kinases in plants J Exp Bot 58 3503-3511 822

Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X and Zhou JM (2008) Firefly 823 luciferase complementation imaging assay for protein-protein interactions in plants 824 Plant Physiol 146368-376 825

Dorjgotov D Jurca ME Fodor-Dunai C Szűcs A Őtvős K Klement Eacute Biacuteroacute J Feheacuter A 826 (2009) Plant Rho-type (Rop) GTPase dependent activation of receptor-like 827 cytoplasmic kinases in vitro FEBS letters 5831175-1182 828

Gampala SS Kim TW He JX Tang WQ Deng Z Bai MY Guan S Lalonde Y Sun Y 829 Gendron JM Chen H Shibagaki N Ferl RJ Ehrhardt D Chong K Burlingame AL 830 and Wang ZY (2007) An Essential Role for 14-3-3 Proteins in Brassinosteroid Signal 831 Transduction in Arabidopsis Developmental Cell 13177-189 832

Gendron JM Liu JS Fan M Bai MY Wenkel S Springer PS Barton MK and Wang ZY 833 (2012) Brassinosteroids regulate organ boundary formation in the shoot apical 834 meristem of Arbidopsis Proc Natl Acad Sci USA 10921152-21157 835

Gomes MMA (2011) Physiological effects related to brassinosteroid application in 836 plants Brassinosteroids A Class of Plant Hormone eds Hayat S Ahmad A 837 (Springer) pp193-242 838

Gruszka D Szarejko I and Maluszynski M (2011) New allele of HvBRI1 gene encoding 839 brassinosteroid receptor in barley J Appl Genetics 52257-268 840

Gruumltter C Sreeramulu S Sessa G Rauh D (2013) Structural Characterization of the 841 RLCK Family Member BSK8 A Pseudokinase with an Unprecedented Architecture 842 Journal of Molecular Biology 4254455-4467 843

Guan SH Price JC Prusiner SB Ghaemmaghami S and Burlingame AL (2011) A data 844 processing pipeline for mammalian proteome dynamics studies using stable isotope 845 metabolic labeling Molecular amp Cellular Proteomics Dec10(12)M111010728 doi 846 101074 847

Hartwig T Chuck GS Fujioke S Klempien A Weizbauer R Potluri DPV Choe S Johal 848 GS Schulz B (2011) Brassinosteroid control of sex determination in maize Proc 849


41

Natl Acad Sci USA 10819814-19819 850 He JX Gendron JM Sun Y Gampala SSL Gendron N Sun CQ Wang ZY (2005) BZR1 851

is a transcriptional repressor with dual roles in brassinosteroid homeostasis and 852 growth response Science 3071634-1638 853

He JX Gendron JM Yang Y Li J and Wang ZY (2002) The GSK3-like kinase BIN2 854 phosphorylates and destabilizes BZR1 a positive regulator of the brassinosteroid 855 signaling pathway in Arabidopsis Proc Natl Acad Sci USA 99 10185-10190 856

Hong Z Ueguchi-Tanaka U and Matsuoka M (2004) Brassinosteroids and rice 857 architecture J Pestic Sci 29184-188 858

Kakita M (2007) Two distinct forms of M-locus protein kinase localize to the plasma 859 membrane and interact directly with S-locus receptor kinases to transduce 860 self-incompatibility signaling in Brassica rapa Plant Cell 19 3961-3973 861

Kim TW Guan SH Burlingame AL Wang ZY (2011) The CDG1 kinase mediates 862 brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase 863 and GSK3-like kinase BIN2 Molecular Cell 43561-571 864

Kim TW Guan SH Sun Y Deng ZP Tang WQ Shang JX Sun Y Burlingame AL Wang 865 ZY (2009) Brassinosteroid signal transduction from cell surface receptor kinases to 866 nuclear transcription factors Nature Cell Biology 111254-U233 867

Kim TW Michniewicz M Bergmann DC Wang ZY (2012) Brassinosteroid regulates 868 stomatal development by GSK3 mediated inhibition of a MAPK pathway Nature 869 482419-U1526 870

Koka CV Cerny RE Gardner RG Noguchi T Fujioka S Takatsuto S Yoshida S and 871 Clouse SD (2000) A putative role for the tomato genes DUMPY and CURL-3 in 872 brassinosteroid biosynthesis and response Plant Phyisiol 12285-98 873

Kutschera U and Wang ZY (2012) Brassinosteroid action in flowering plants a 874 Darwinian perspective J Exp Bot 875

Li JM and Chory J (1997) A putative leucine-rice repeat receptor kinase involved in 876 brassinosteroid signal transduction Cell 90929-938 877

Li D Wang L Wang M Xu YY Luo W Liu YJ Xu ZH Li J Chong K (2009) 878 Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high 879 yield Plant Biotechnol J 7791-806 880

Li J Wen J Lease KA Doke JT Tas FE and Walker JC (2002) BAK1 an Arabidopsis 881 LRR receptor-like protein kinase interacts with BRI1 and modulates brassinosteroid 882 signaling Cell 110213-222 883

Liu J Zhong S Guo X Hao L Wei X Huang Q Hou Y Shi J Wang C Gu H and Qu LJ 884 (2013) Membrane-bound RLCKs LIP1 and LIP2 are essential male factors 885 controlling male-female attraction in Arabidopsis Current Biology 23993-998 886

Lu D Wu S Gao X Zhang Y Shan L and He P (2010) A receptor-like cytoplasmic kinase 887 BIK1 associates with a flagellin receptor complex to initiate plant innate immunity 888 Proc Natl Acad Sci USA 107 496-501 889

Muto H Yabe N Asami T Hasunuma K and Yamamoto KT (2004) Overexpression of 890


42

constitutive differential growth 1 gene which encodes a RLCKVII-subfamily protein 891 kinase causes abnormal differential and elongation growth after organ differentiation 892 in Arabidopsis Plant Physiol 136 3124-3133 893

Nakamura A Fujioka S Sunohar H Kamiya N Hong Z Inukai Y Miura K Takatsuto S 894 Yoshida S Ueguchi-tanaka M Hasegawa Y Kitano H and Matsuoka (2006) The 895 role of OsBRI1 and its homologous genes OsBRL1 and OsBRL3 in rice Plant 896 Physiol 140580-590 897

Nam KH and Li J (2002) BRI1BAK1 a receptor kinase pair mediating brassinosteroid 898 signaling Cell 110203-212 899

Nomura T Bishop GJ Kaneta T Reid JB Chory J and Yokota T (2003) The LKA gene is 900 a BRASSINOSTEROID INSENSITIVE 1 homolog of pea Plant J 36291-300 901

Roux F Noel L Rivas S and Roby D (2014) ZRK atypical kinases emerging signaling 902 components of plant immunity New Phytologist 203713-716 903

Santiago J Henzler C and Hothorn M (2013) Molecular Mechanism for Plant Steroid 904 Receptor Activation by Somatic Embryogenesis Co-Receptor Kinases Science 905 341889-892 906

Sreeramulu S Mostizky Y Sunitha S Shani E Nahum H Salomon D Ben HL Gruetter 907 C Rauh D Ori N and Sessa G (2013) BSKs are partially redundant positive 908 regulators of brassinosteroid signaling in Arabidopsis Plant J 74905-919 909

Shi H Shen Q Qi Y Yan H Nie H Chen Y Zhao T Katagiri F and Tang D (2013) 910 BR-SIGNALING KINASE1 Physically Associates with FLAGELLIN SENSING2 911 and Regulates Plant Innate Immunity in Arabidopsis Plant Cell 25 1143-1157 912

Sun Y Fan XY Cao DM Tang WQ He K Zhu JY He JX Bai MY Zhu SW Oh E Patil 913 S Kim TW Ji HK Wong WH Rhee SY Wang ZY (2010) Integration of 914 Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth 915 Regulation in Arabidopsis Dev Cell 19765-777 916

Sun Y Han Z Tang J Hu Z Chai C Zhou B Chai J (2013) Structure reveals that BAK1 917 as a co-receptor recognizes the BRI1-bound brassinolide Cell Res 231326-1329 918

Tang W (2012) Quantitative analysis of plasma membrane proteome using 919 two-dimensional difference gel electrophoresis Methods in Molecular Biology 920 87667-82 921

Tang W Kim T Oses-Prieto JA Sun Y Deng Z Zhu S Wang R Burlingame AL Wang 922 ZY (2008) BSKs mediate signal transduction from the receptor kinase BRI1 in 923 Arabidopsis Science 321 557-560 924

Tang W Yuan M Wang RJ Yang YH Wang CM Oses-Prieto JA Kim TW Zhou HW 925 Deng ZP Gampala SS Gendron JM Jonassen EM Lillo C Delong A Burlingame 926 AL Sun Y Wang ZY (2011) PP2A activates brassinosteroid-responsive gene 927 expression and plant growth by dephosphorylating BZR1 Nature Cell Biology 928 13124-131 929

Thompson GA and Okuyama H (2000) Lipid-linked proteins of plants Progress in lipid 930 research 39(1) 19-39 931


43

Tong HN Liu LC Jin Y Du L Yin YH Qian Q Zhu LH Chu CC (2012) DWARF AND 932 LOW-TILLERING Acts as a Direct Downstream Target of a GSK3SHAGGY-Like 933 Kinase to Mediate Brassinosteroid Responses in Rice Plant Cell 24 2562-2577 934

Vert G Chory J (2006) Downstream nuclear events in brassinosteroid signaling Nature 935 44196-100 936

Vriet C Russinova E Reuzeau C (2012) Boosting Crop Yields with Plant Steroids 937 The Plant Cell 24 842-857 938

Wang XF Goshe MB Soderblom EJ Phinney BS Kuchar JA Li J Asami T Yoshida S 939 Huber SC Clouse SD (2005) Identification and functional analysis of in vivo 940 phosphorylation sites of the Arabidopsis BRASSINOSTEROID INSENSITIVE1 941 receptor kinase Plant Cell 171685-1703 942

Wang XF Kota U He K Blackburn K Li J Goshe MB Huber SC Clouse SD (2008) 943 Sequential transphosphorylation of the BRI1BAK1 receptor kinase complex impacts 944 early events in brassinosteroid signaling Developmental Cell 15220-235 945

Wu X Oh MH Kim HS Schwartz D Imai BS Yau PM Clouse SD and Huber SC 946 (2012) Transphosphorylation of E coli proteins during production of recombinant 947 protein kinases provides a robust system to characterize kinase specificity Frontiers 948 in Plant Science 3262 949

Yamaguchi K Yamada K Ishikawa K Yoshimura S Hayashi N Uchihashi K Ishihama 950 N Kishi-Kaboshi M Takahashi A Tsuge S Ochiai H Tada Y Shimamoto K 951 Yoshioka H Kawasaki T (2013) A receptor-like cytoplasmic kinase targeted by a 952 plant pathogen effector is directly phosphorylated by the chitin receptor and mediates 953 rice immunity Cell Host Microbe 13 343-357 954

Yang Y Peng H Huang H Wu J Jia S Huang D Lu T (2004) Large-scale 955 production of enhancer trapping lines for rice functional genomics Plant Science 956 167281-288 957

Yin Y Vafeados D Tao Y Yoshida S Asami T and Chory J (2005) A new class of 958 transcription factors mediates brassinosteroid-regulated gene expression in 959 Arabidopsis Cell 120249-259 960

Zhang J Li W Xiang T Liu Z Laluk K Ding X Zou Y Gao M Zhang X Chen S 961 Mengiste T Zhang Y and Zhou JM (2010) Receptor-like cytoplasmic kinases 962 integrate signaling from multiple plant immune receptors and are targeted by a 963 pseudomonas syringae effector Cell Host Microbe 7290-301 964

965


Parsed CitationsAbuQamar S Chai MF Luo H Song F and Mengiste T (2008) Tomato protein kinase 1b mediates signaling of plant responses tonecrotrophic fungi and insect herbivory Plant Cell 201964-1983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Allan RK and Ratajczak T (2011) Versatile TPR domains accommodate different modes of target protein recognition and functionCell Stress and Chaperones 16353-367


Bai MY Zhang LY Gampala SS Zhu SW Song WY Chong K and Wang ZY (2007) Functions of OsBZR1 and 14-3-3 proteins inbrassinosteroid signaling in rice Proc Natl Acad Sci USA 10413839-13844


Burr CA Leslie ME Orlowski SK Chen I Wright CE Daniels MJ And Liljegren SJ (2011) CAST AWAY a membrane associatedreceptor-like kinase inhibits organ abscission in Arabidopsis Plant Physiology 156 1837-1850


Castelles E and Casacuberta JM (2007) Signalling through kinase defective domains the prevalence of atypical receptor-likekinases in plants J Exp Bot 58 3503-3511


Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X and Zhou JM (2008) Firefly luciferase complementation imaging assay forprotein-protein interactions in plants Plant Physiol 146368-376


Dorjgotov D Jurca ME Fodor-Dunai C Szucs A Otvos K Klement Eacute Biacuteroacute J Feheacuter A (2009) Plant Rho-type (Rop) GTPasedependent activation of receptor-like cytoplasmic kinases in vitro FEBS letters 5831175-1182


Gampala SS Kim TW He JX Tang WQ Deng Z Bai MY Guan S Lalonde Y Sun Y Gendron JM Chen H Shibagaki N Ferl RJEhrhardt D Chong K Burlingame AL and Wang ZY (2007) An Essential Role for 14-3-3 Proteins in Brassinosteroid SignalTransduction in Arabidopsis Developmental Cell 13177-189


Gendron JM Liu JS Fan M Bai MY Wenkel S Springer PS Barton MK and Wang ZY (2012) Brassinosteroids regulate organboundary formation in the shoot apical meristem of Arbidopsis Proc Natl Acad Sci USA 10921152-21157


Gomes MMA (2011) Physiological effects related to brassinosteroid application in plants Brassinosteroids A Class of PlantHormone eds Hayat S Ahmad A (Springer) pp193-242


Gruszka D Szarejko I and Maluszynski M (2011) New allele of HvBRI1 gene encoding brassinosteroid receptor in barley J ApplGenetics 52257-268


Gruumltter C Sreeramulu S Sessa G Rauh D (2013) Structural Characterization of the RLCK Family Member BSK8 A Pseudokinasewith an Unprecedented Architecture Journal of Molecular Biology 4254455-4467


Guan SH Price JC Prusiner SB Ghaemmaghami S and Burlingame AL (2011) A data processing pipeline for mammalian proteomedynamics studies using stable isotope metabolic labeling Molecular amp Cellular Proteomics Dec10(12)M111010728 doi 101074

Pubmed Author and TitleCrossRef Author and Title


Google Scholar Author Only Title Only Author and Title

Hartwig T Chuck GS Fujioke S Klempien A Weizbauer R Potluri DPV Choe S Johal GS Schulz B (2011) Brassinosteroidcontrol of sex determination in maize Proc Natl Acad Sci USA 10819814-19819


He JX Gendron JM Sun Y Gampala SSL Gendron N Sun CQ Wang ZY (2005) BZR1 is a transcriptional repressor with dual rolesin brassinosteroid homeostasis and growth response Science 3071634-1638


He JX Gendron JM Yang Y Li J and Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1 apositive regulator of the brassinosteroid signaling pathway in Arabidopsis Proc Natl Acad Sci USA 99 10185-10190


Hong Z Ueguchi-Tanaka U and Matsuoka M (2004) Brassinosteroids and rice architecture J Pestic Sci 29184-188Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kakita M (2007) Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-locusreceptor kinases to transduce self-incompatibility signaling in Brassica rapa Plant Cell 19 3961-3973


Kim TW Guan SH Burlingame AL Wang ZY (2011) The CDG1 kinase mediates brassinosteroid signal transduction from BRI1receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2 Molecular Cell 43561-571


Kim TW Guan SH Sun Y Deng ZP Tang WQ Shang JX Sun Y Burlingame AL Wang ZY (2009) Brassinosteroid signaltransduction from cell surface receptor kinases to nuclear transcription factors Nature Cell Biology 111254-U233


Kim TW Michniewicz M Bergmann DC Wang ZY (2012) Brassinosteroid regulates stomatal development by GSK3 mediatedinhibition of a MAPK pathway Nature 482419-U1526


Koka CV Cerny RE Gardner RG Noguchi T Fujioka S Takatsuto S Yoshida S and Clouse SD (2000) A putative role for thetomato genes DUMPY and CURL-3 in brassinosteroid biosynthesis and response Plant Phyisiol 12285-98


Kutschera U and Wang ZY (2012) Brassinosteroid action in flowering plants a Darwinian perspective J Exp BotPubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Li JM and Chory J (1997) A putative leucine-rice repeat receptor kinase involved in brassinosteroid signal transduction Cell90929-938


Li D Wang L Wang M Xu YY Luo W Liu YJ Xu ZH Li J Chong K (2009) Engineering OsBAK1 gene as a molecular tool toimprove rice architecture for high yield Plant Biotechnol J 7791-806


Li J Wen J Lease KA Doke JT Tas FE and Walker JC (2002) BAK1 an Arabidopsis LRR receptor-like protein kinase interactswith BRI1 and modulates brassinosteroid signaling Cell 110213-222


Liu J Zhong S Guo X Hao L Wei X Huang Q Hou Y Shi J Wang C Gu H and Qu LJ (2013) Membrane-bound RLCKs LIP1 andLIP2 are essential male factors controlling male-female attraction in Arabidopsis Current Biology 23993-998

Pubmed Author and Title httpsplantphysiolorgDownloaded on December 15 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Lu D Wu S Gao X Zhang Y Shan L and He P (2010) A receptor-like cytoplasmic kinase BIK1 associates with a flagellin receptorcomplex to initiate plant innate immunity Proc Natl Acad Sci USA 107 496-501


Muto H Yabe N Asami T Hasunuma K and Yamamoto KT (2004) Overexpression of constitutive differential growth 1 gene whichencodes a RLCKVII-subfamily protein kinase causes abnormal differential and elongation growth after organ differentiation inArabidopsis Plant Physiol 136 3124-3133


Nakamura A Fujioka S Sunohar H Kamiya N Hong Z Inukai Y Miura K Takatsuto S Yoshida S Ueguchi-tanaka M Hasegawa YKitano H and Matsuoka (2006) The role of OsBRI1 and its homologous genes OsBRL1 and OsBRL3 in rice Plant Physiol140580-590


Nam KH and Li J (2002) BRI1BAK1 a receptor kinase pair mediating brassinosteroid signaling Cell 110203-212Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Nomura T Bishop GJ Kaneta T Reid JB Chory J and Yokota T (2003) The LKA gene is a BRASSINOSTEROID INSENSITIVE 1homolog of pea Plant J 36291-300


Roux F Noel L Rivas S and Roby D (2014) ZRK atypical kinases emerging signaling components of plant immunity NewPhytologist 203713-716


Santiago J Henzler C and Hothorn M (2013) Molecular Mechanism for Plant Steroid Receptor Activation by SomaticEmbryogenesis Co-Receptor Kinases Science 341889-892


Sreeramulu S Mostizky Y Sunitha S Shani E Nahum H Salomon D Ben HL Gruetter C Rauh D Ori N and Sessa G (2013) BSKsare partially redundant positive regulators of brassinosteroid signaling in Arabidopsis Plant J 74905-919


Shi H Shen Q Qi Y Yan H Nie H Chen Y Zhao T Katagiri F and Tang D (2013) BR-SIGNALING KINASE1 Physically Associateswith FLAGELLIN SENSING2 and Regulates Plant Innate Immunity in Arabidopsis Plant Cell 25 1143-1157


Sun Y Fan XY Cao DM Tang WQ He K Zhu JY He JX Bai MY Zhu SW Oh E Patil S Kim TW Ji HK Wong WH Rhee SY WangZY (2010) Integration of Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth Regulation inArabidopsis Dev Cell 19765-777


Sun Y Han Z Tang J Hu Z Chai C Zhou B Chai J (2013) Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide Cell Res 231326-1329


Tang W (2012) Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresisMethods in Molecular Biology 87667-82


Tang W Kim T Oses-Prieto JA Sun Y Deng Z Zhu S Wang R Burlingame AL Wang ZY (2008) BSKs mediate signal transductionfrom the receptor kinase BRI1 in Arabidopsis Science 321 557-560

Pubmed Author and TitlehttpsplantphysiolorgDownloaded on December 15 2020 - Published by

Copyright (c) 2020 American Society of Plant Biologists All rights reserved


Tang W Yuan M Wang RJ Yang YH Wang CM Oses-Prieto JA Kim TW Zhou HW Deng ZP Gampala SS Gendron JM JonassenEM Lillo C Delong A Burlingame AL Sun Y Wang ZY (2011) PP2A activates brassinosteroid-responsive gene expression andplant growth by dephosphorylating BZR1 Nature Cell Biology 13124-131


Thompson GA and Okuyama H (2000) Lipid-linked proteins of plants Progress in lipid research 39(1) 19-39Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Tong HN Liu LC Jin Y Du L Yin YH Qian Q Zhu LH Chu CC (2012) DWARF AND LOW-TILLERING Acts as a Direct DownstreamTarget of a GSK3SHAGGY-Like Kinase to Mediate Brassinosteroid Responses in Rice Plant Cell 24 2562-2577


Vert G Chory J (2006) Downstream nuclear events in brassinosteroid signaling Nature 44196-100Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Vriet C Russinova E Reuzeau C (2012) Boosting Crop Yields with Plant Steroids The Plant Cell 24 842-857Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Wang XF Goshe MB Soderblom EJ Phinney BS Kuchar JA Li J Asami T Yoshida S Huber SC Clouse SD (2005) Identificationand functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID INSENSITIVE1 receptor kinasePlant Cell 171685-1703


Wang XF Kota U He K Blackburn K Li J Goshe MB Huber SC Clouse SD (2008) Sequential transphosphorylation of theBRI1BAK1 receptor kinase complex impacts early events in brassinosteroid signaling Developmental Cell 15220-235


Wu X Oh MH Kim HS Schwartz D Imai BS Yau PM Clouse SD and Huber SC (2012) Transphosphorylation of E coli proteinsduring production of recombinant protein kinases provides a robust system to characterize kinase specificity Frontiers in PlantScience 3262


Yamaguchi K Yamada K Ishikawa K Yoshimura S Hayashi N Uchihashi K Ishihama N Kishi-Kaboshi M Takahashi A Tsuge SOchiai H Tada Y Shimamoto K Yoshioka H Kawasaki T (2013) A receptor-like cytoplasmic kinase targeted by a plant pathogeneffector is directly phosphorylated by the chitin receptor and mediates rice immunity Cell Host Microbe 13 343-357


Yang Y Peng H Huang H Wu J Jia S Huang D Lu T (2004) Large-scale production of enhancer trapping lines for ricefunctional genomics Plant Science 167281-288


Yin Y Vafeados D Tao Y Yoshida S Asami T and Chory J (2005) A new class of transcription factors mediatesbrassinosteroid-regulated gene expression in Arabidopsis Cell 120249-259


Zhang J Li W Xiang T Liu Z Laluk K Ding X Zou Y Gao M Zhang X Chen S Mengiste T Zhang Y and Zhou JM (2010) Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a pseudomonas syringaeeffector Cell Host Microbe 7290-301



Parsed Citations

Reviewer PDF

Parsed Citations

2

OsBRI1 activates BR signaling by preventing binding between the TPR and kinase 18

domains of OsBSK3 via phosphorylation 19

20

Baowen Zhang1 Xiaolong Wang1 Zhiying Zhao1 Ruiju Wang1 Xiahe Huang2 Yali Zhu1 21

Li Yuan1 Yingchun Wang2 Xiaodong Xu1 Alma L Burlingame3 Yingjie Gao1 Yu Sun1 22

and Wenqiang Tang1 23

24

25

1 Key Laboratory of Molecular and Cellular Biology of Ministry of Education Hebei 26

Collaboration Innovation Center for Cell Signaling Hebei Key Laboratory of 27

Molecular and Cellular Biology College of Life Sciences Hebei Normal University 28

Shijiazhuang 050016 China 29

2 Key Laboratory of Molecular Development Biology Insitute of Genetics and 30

Development Biology Chinese Academy of Sciences Beijing 100101 China 31

3 Mass spectrometry facility Department of Pharmaceutical Chemistry University of 32

California San Francisco California 94143 33

34

35

36

37

38

One-sentence summary 39

OsBRI1 disrupts the interaction between the TPR and kinase domains of OsBSK3 via 40

phosphorylation this activates BR signaling by increasing the binding between OsBSK3rsquos 41

kinase domain and BSU1 42

43

44


3






50


52











63

64

65


4

ABSTRACT 66






5














kinase BRI1 84

85


6

INTRODUCTION 86




























7


























OsBSK3 and BSU1 138

139


8

RESULTS 140






















in Arabidopsis 162





9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

3






50


52











63

64

65


4

ABSTRACT 66






5














kinase BRI1 84

85


6

INTRODUCTION 86




























7


























OsBSK3 and BSU1 138

139


8

RESULTS 140






















in Arabidopsis 162





9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

4

ABSTRACT 66






5














kinase BRI1 84

85


6

INTRODUCTION 86




























7


























OsBSK3 and BSU1 138

139


8

RESULTS 140






















in Arabidopsis 162





9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

5














kinase BRI1 84

85


6

INTRODUCTION 86




























7


























OsBSK3 and BSU1 138

139


8

RESULTS 140






















in Arabidopsis 162





9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

6

INTRODUCTION 86




























7


























OsBSK3 and BSU1 138

139


8

RESULTS 140






















in Arabidopsis 162





9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

7


























OsBSK3 and BSU1 138

139


8

RESULTS 140






















in Arabidopsis 162





9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

8

RESULTS 140






















in Arabidopsis 162





9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

9
















10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

10
















11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

11















12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

12




























13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

13





























14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

14





























15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

15















16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

16





and S12A) 304




17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

17




























18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

18






19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

19

























rice 361




20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

20










373


21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

21

DISCUSSION 374




























22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

22





























23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

23





























24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

24























477




25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

25




























26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

26





























27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

27




























28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

28




























29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

29
























spectrometry 608




30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

30





























31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

31








645 646


32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

32

















673


33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

33


[M+H]+ Calculated




34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

34

Acknowledgement 679


682


35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

35

FIGURE LEGENDS 683










693













706





36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

36








717





















37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

37

737












749
















38






(Plt005) 769

770






















39





795













808


40

















41


















42
















43











965










































































































Parsed Citations

Reviewer PDF

Parsed Citations

38






(Plt005) 769

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Running title: Negative regulation of OsBSK3 via its …...2015/12/22 · 21 Baowen Zhang1,...

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